Methods for improved diagnosis and treatment of mycobacterial infections

ABSTRACT

Media for growth enhancement and resuscitation of mycobacteria (such as  Mycobacterium tuberculosis, Mycobacterium paratuberculosis , and  Mycobacterium leprae ) are provided. The media comprise isolated cell extract, early-stationary-phase or stationary phase supernatant, or a substantially purified component thereof such as a protein, a peptide fragment of the protein, or a phospholipid. The protein is Rv1147c and the phospholipid or a component of a phospholipid. Diagnostic methods and kits utilizing the media are also provided, as well as treatment methods utilizing spent culture supernatant and cell extracts, or components thereof.

[0001] This invention was made using funds from the National Institutesof Health having grant number A140584. The government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention generally relates to improved diagnosis andtreatment of mycobacterial infections. In particular, the inventionprovides methods for using spent culture supernatant, components of thespent culture supernatant, or cell extracts to enhance cultivation ofmycobacteria, or to revive dormant mycobacteria bacilli.

[0004] 2. Background of the Invention

[0005] Tuberculosis (TB) is a leading infectious killer worldwide with 8million new cases and 2 million deaths a year (1). One third of theworld population is latently infected with Mycobacterium tuberculosis.The success of M. tuberculosis as a human pathogen relates to itsremarkable ability to persist for long periods of time in the face ofimmunity and chemotherapy.

[0006] The current TB treatment is suboptimal, requiring a minimum of 6months using the WHO recommended treatment regimen (DOTS, DirectlyObserved Treatment, Shortcourse), which consists of 4 drugs isoniazid,rifampin, pyrazinamide and ethambutol used in combination (WHO Report onthe Tuberculosis Epidemic, 2000). Such lengthy treatments are expensive,and are likely to be problematic in terms of patient compliance. Inaddition, one potentially catastrophic effect of the lengthy therapy isthe development of drug-resistant TB.

[0007] This lengthy treatment is thought to be due to the presence of apopulation of dormant bacilli in vivo that are not effectively killed bycurrent TB drugs (McKinney et al., 1998). Dormant bacilli can bedemonstrated in the Cornell mouse model of dormancy (McCune et al,1966), where mice infected with tubercle bacilli were treated with INHand PZA for 2 months, at which time no viable bacilli were demonstrablein the tissues as judged by colony forming units (CFU); yet diseaserelapsed with viable yet drug susceptible bacilli after cessation oftreatment for 3 months in one third of mice or in almost all mice givenimmunosuppressing steroids. This suggests that the drugs are unable toeliminate dormant bacilli completely and that although the dormantbacilli do not form colonies on plates they are not dead and can reviveand cause disease when the immune system is compromised.

[0008] The unresponsiveness of dormant or nongrowing bacilli to DOTS isphenotypic or physiologic but not genetic, so that when the dormantbacilli revive and start growing they become susceptible to TB drugsagain. Therefore, agents that cause dormant bacilli to revive andresuscitate so that they respond to treatment are potential modulatorsof drug activity in the host and can be used for improved treatment ofthe disease by potentially shortening the treatment time.

[0009] The current diagnosis of tuberculosis still relies on culture ofthe M. tuberculosis organism as the definitive method of diagnosis.However, M. tuberculosis grows very slowly and it takes several weeksfor the primary isolation of the bacilli from clinical specimens forconfirmation of the disease. Current clinical diagnosis uses solid mediasuch as Lowenstein-Jensen medium, 7H10 or 7H11 agar medium and liquid7H12B medium as in BACTEC460 machine for primary isolation of thebacilli from clinical specimen. In general, the liquid 7H12B basedmedium is more sensitive in terms of primary isolation of positivecultures from clinical specimens. However, the current medium forisolation of M. tuberculosis from clinical specimens is not optimal.Even with liquid 7H12B medium in the presence of growth enhancing agentPOES (polyoxyethelene stearate) (Becton Dickinson, Sparks, Md., U.S.Pat. No. 4,769,332), the isolation rate is about 80%, and some 20%samples which later prove to be containing the bacilli are not easilydetected. Agents that can improve the primary isolation sensitivity andenhance the growth of M tuberculosis should improve the ability todiagnose TB.

SUMMARY OF THE INVENTION

[0010] The present invention provides media and methods for enhancingthe cultivation of mycobacteria, or reviving (resuscitating) dormantbacilli from mycobacterium species. The media and methods utilizemycobacterial products from the M. tuberculosis complex. By “M.tuberculosis complex” we mean M. tuberculosis complex organisms whichinclude M. tuberculosis, Mycobacterium bovis, including the vaccinestrain BCG, and Microbacterium microti. The products include cellextracts, early-stationary-phase culture supernatant (ESPSN), andstationary phase culture supernatant (SPSN), either crude or assubstantially purified components from these sources. The products maybe used to enhance the growth of mycobacterial species that aredifficult to culture, and/or to effect the resuscitation of dormantmycobacteria bacilli.

[0011] It is an object of this invention to provide a supplementedmedium for culturing mycobacterium species, for example Mycobacteriumtuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae.The medium comprises a cell extract from M. tuberculosis complex, or atleast one product from M. tuberculosis complex (e.g. a component ofearly-stationary-phase culture supernatant (ESPSN), of stationary phaseculture supernatant (SPSN), or of a cell extract) and a suitable culturemedium. The cell extract or substantially purified product exhibitsresuscitation activity for dormant bacilli of the mycobacterium species,or the ability to enhance the growth of the mycobacterium species. Thesubstantially purified product may be a phospholipid or a component of aphospholipid, such as phosphotidyl-L-serine, dioleoylphosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine,tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively,the substantially purified product may be a protein or a fragment of aprotein, e.g. protein Rv1147c (accession number F70875), a peptidecorresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2,a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQID NO. 4 and a peptide corresponding to SEQ ID NO. 5.) Suitable culturemedia to be supplemented include 7H12B, 7H9, 7H10, 7H11, Sauton'smedium, Dubos medium, and egg-based media (for example Lowenstein-Jensenmedium). In addition, a mixture of substantially purified component ofESPSN or SPSN may be utilized, e.g. a mixture of phospholipids and/orproteins and peptides.

[0012] In addition, isolated and sterilized ESPSN or SPSN of M.tuberculosis complex can itself be utilized as a culture medium, orcombined with a suitable fresh culture medium or other fresh nutrientsto produce supplemented culture medium. Further, substantially purifiedcomponents of ESPSN, SPSN, or mycobacterium cell extracts thatresuscitation activity for dormant bacilli of the mycobacterium speciesmay be added.

[0013] In another aspect, the present invention provides a method forreviving dormant mycobacterium bacilli of, for example Mycobacteriumtuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae.According to the method, dormant bacilli are exposed to a cell extractor at least one substantially purified product of M tuberculosis complex(e.g. a component of early-stationary-phase culture supernatant (ESPSN),of stationary phase culture supernatant (SPSN), or of a cell extract).The cell extract and substantially purified product exhibitresuscitation activity for dormant bacilli of the mycobacterium species,and the cell extract or product is present in sufficient quantity toeffect revival of the dormant bacilli. The substantially purifiedproduct may be a phospholipid or component thereof, e.g.phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine,phosphotidylcholine, or phosphotidylethanolamine, tuberculostearic acid,arachidonic acid, and fatty acids.

[0014] Alternatively, the substantially purified product may be aprotein or a fragment of a protein, e.g., protein Rv1147c, a peptidecorresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2,a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQID NO. 4 and a peptide corresponding to SEQ ID NO. 5.) In addition, amixture of substantially purified products may be utilized, e.g. amixture of phospholipids and/or proteins and peptides.

[0015] In addition, dormant bacilli may be revived by exposure to cellextract or isolated and sterilized ESPSN or SPSN of M. tuberculosiscomplex, which may also be combined with a suitable fresh culture mediumor other fresh nutrients (e.g. substantially purified components ofESPSN, SPSN, or mycobacterium cell extracts that exhibit resuscitationactivity for dormant bacilli).

[0016] In yet another aspect of the present invention, a method for thediagnosis of an infection caused by a mycobacterium species (for exampleMycobacterium tuberculosis, Mycobacterium paratuberculosis, andMycobacterium leprae) is provided. The method comprises combining asample for which the presence or absence of the mycobacterium species isto be determined with medium supplemented with cell extract or at leastone substantially purified product M. tuberculosis complex (e.g. acomponent of early-stationary-phase culture supernatant (ESPSN), ofstationary phase culture supernatant (SPSN), or of a cell extract); andanalyzing the culture for the presence of the mycobacterium species. Thecell extract and substantially purified product exhibits growthenhancement and/or resuscitation activity for dormant bacilli of themycobacterium species. If the mycobacterium species is found in theculture, this indicates a positive diagnosis for the infection. Thesubstantially purified product may be a phospholipid or component of aphospholipid, e.g. phosphotidyl-L-serine, dioleoylphosphotidyl-L-serine, phosphotidylcholine, or phosphotidylethanolamine,tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively,the substantially purified product may be a protein or a fragment of aprotein, e.g. protein Rv1147c, a peptide corresponding to SEQ ID NO. 1,a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQID NO. 3, a peptide corresponding to SEQ ID NO. 4 and a peptidecorresponding to SEQ ID NO. 5.) In a ddition, a mixture of substantiallypurified products may be utilized, e.g. a mixture of phospholipidsand/or proteins and peptides.

[0017] In addition, the method for the diagnosis of an infection causedby a mycobacterium species may comprises combining a sample for whichthe presence or absence of the mycobacterium species is to be determinedwith isolated and sterilized ESPSN or SPSN of M. tuberculosis complex,which may also be combined with a suitable fresh culture medium or otherfresh nutrients (e.g. substantially purified components of ESPSN, SPSN,or mycobacterium cell extracts that growth enhancing or resuscitationactivity for dormant bacilli).

[0018] In another aspect of the present invention, a kit for thediagnosis of an infection caused by a mycobacterium species (for exampleMycobacterium tuberculosis, Mycobacterium paratuberculosis, andMycobacterium leprae) is provided. The kit includes a sealed containerof medium supplemented with cell extract or at least one substantiallypurified product of M. tuberculosis complex (e.g. a component ofearly-stationary-phase culture supernatant (ESPSN), of stationary phaseculture supernatant (SPSN), or of a cell extract), which may furthercomprise additional fresh media or nutrients. The cell extract orsubstantially purified product exhibits growth enhancement and/orresuscitation activity for dormant bacilli of the mycobacterium species.The substantially purified product may be a phospholipid or a componentof a phospholipid e.g. phosphotidyl-L-serine, dioleoylphosphotidyl-L-serine, phosphotidylcholine, or phosphotidylethanolamine,tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively,the substantially purified product may be a protein or a fragment of aprotein, e.g. protein Rv1147c, a peptide corresponding to SEQ ID NO. 1,a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQID NO. 3, a peptide corresponding to SEQ ID NO. 4 and a peptidecorresponding to SEQ ID NO. 5.) In addition, a mixture of substantiallypurified products may be utilized, e.g. a mixture of phospholipidsand/or proteins and peptides.

[0019] In addition, the kit may comprise isolated and sterilized ESPSNor SPSN of M. tuberculosis complex, which may also be combined with asuitable fresh culture medium or other fresh nutrients (e.g.substantially purified components of ESPSN, SPSN, or mycobacterium cellextracts that exhibit growth enhancement or resuscitation activity fordormant bacilli).

[0020] The present invention further provides a method for the treatmentof an infection caused by a mycobacterium species (for exampleMycobacterium tuberculosis, Mycobacterium paratuberculosis, andMycobacterium leprae). According to the method, cell extract or at leastone substantially purified product of M. tuberculosis complex (e.g. acomponent of early-stationary-phase culture supernatant (ESPSN), ofstationary phase culture supernatant (SPSN), or of a cell extract), isadministered to said patient, in conjunction with drugs of anestablished treatment protocol for the infection in order to amelioratesymptoms associated with the infection. Administering such a substanceresults in the revival of dormant bacilli of the mycobacterium speciesin the patient, thus making the bacilli susceptible to treatment with anantibiotic. (Dormant bacilli are otherwise not susceptible to currentdrug therapy protocols.) The substantially purified product may be aphospholipid e.g. phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine,phosphotidylcholine, or phosphotidylethanolamine, tuberculostearic acid,arachidonic acid, and fatty acids. Alternatively, the substantiallypurified product may be a protein or a fragment of a protein, e.g.protein Rv1147c, a peptide corresponding to SEQ ID NO. 1, a peptidecorresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3,a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding toSEQ ID NO. 5.) In addition, a mixture of substantially purified productsmay be utilized, e.g. a mixture of phospholipids and/or proteins andpeptides.

[0021] In another aspect, the present invention provides apharmacological agent for the treatment of an infection caused by amycobacterium species (for example Mycobacterium tuberculosis,Mycobacterium paratuberculosis, and Mycobacterium leprae). The agentcomprises cell extract or at least one substantially purified product ofM. tuberculosis complex (e.g. a component of early-stationary-phaseculture supernatant (ESPSN), of stationary phase culture supernatant(SPSN), or of a cell extract). The substantially purified product may bea phospholipid, e.g. phosphotidyl-L-serine, dioleoylphosphotidyl-L-serine, phosphotidylcholine, or phosphotidylethanolamine,tuberculostearic acid, arachidonic acid, and fatty acids.

[0022] Alternatively, the substantially purified product may be aprotein or a fragment of a protein, (for Mycobacterium tuberculosis,protein Rv1147c, a peptide corresponding to SEQ ID NO. 1, a peptidecorresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3,a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding toSEQ ID NO. 5.), and a physiologically suitable carrier. In addition, amixture of substantially purified products may be utilized, e.g. amixture of phospholipids and/or proteins and peptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0023] The present invention provides media and methods for enhancingthe cultivation of mycobacteria, or reviving (resuscitating) dormantbacilli from mycobacterium species. The media and methods utilizemycobacterial products from, for example, the early stationary phasesupernatant (ESPSN), the stationary phase supernatant (SPSN), or cellextracts of cultures of mycobacterium. The products exhibit growthenhancement of mycobacteria, or resuscitation activity for dormantbacilli of mycobacterium species, examples of which include but are notlimited to Mycobacterium tuberculosis, Mycobacterium paratuberculosis,and Mycobacterium leprae. By the “revival” or “resuscitation” of dormantbacilli, we mean that bacilli that do not display characteristics ofgrowth (e.g. they do not form colonies on suitable media, or do not testas live with FDA-EB staining) obtain the ability to displaygrowth-related characteristics, (e.g. the ability to form colonies onsuitable media or to test as live with FDA-EB staining). By “enhancementof growth” we mean that the amount of growth exhibited by amycobacterial culture is increased at least about two-fold compared to acontrol culture to which the supplement has not been added.

[0024] The invention is predicated on the novel finding that the earlystationary phase supematant (ESPSN) and the stationary phase supernatant(SPSN) of cultures of the mycobacterium have the ability to cause therevival or resuscitation of dormant bacilli, and to generally enhancethe growth of mycobacterial cultures.

[0025] When a batch culture of bacteria grows to stationary phase,bacterial growth halts because of the exhaustion of essential nutrientsand accumulation of toxic products (Postgate, 1967). Upon extendedincubation in stationary phase, bacteria begin to die and viability ofthe culture decreases. The number of colony forming units (CFU) of anaged stationary batch culture is often orders of magnitude less than thetotal number of bacteria in the culture (Amann et al., 1995; Kaprelyantsand Kell, 1993; Votyakova et al., 1994). The nonculturable bacterialpopulation consists of dead cells as well as injured or dormant cells.The present invention capitalizes on the discovery that dormant M.tuberculosis cells can be induced to revive upon exposure to ESPSN orSPSN. Further, individual components of the ESPSN and SPSN that possessthis ability have been identified and substantially purified. Severalembodiments of this discovery are herein disclosed, namely media forculturing mycobacterium, methods for reviving dormant mycobacteriumbacilli, improved methods for the diagnosis of infections caused bymycobacterium, kits for the diagnosis of infections caused bymycobacterium, improved methods for the treatment of infections causedby mycobacterium, and a pharmaceutical preparation for the treatment ofinfections caused by mycobacterium.

[0026] The present invention provides media for culturing amycobacterium species comprising the isolated and sterilized ESPSN orSPSN of the mycobacterium species. In a preferred embodiment of thepresent invention, the mycobacterium species is Mycobacteriumtuberculosis (M. tuberculosis). Those of skill in the art willrecognize, however, that the compositions and methods disclosed hereinare equally applicable to other mycobacterial species, including but notlimited to Mycobacterium paratuberculosis (the causative agent ofCrohn's disease in humans and Johne's disease in cattle), andMycobacterium leprae (the causative agent of leprosy). Mycobacterialspecies have many commonalities, such as the difficulty of establishingthem in culture. It is well known that to initiate the growth of M.tuberculosis, a large inocula are needed and small inocula often fail toinitiate the growth. It is very likely that growth of M. tuberculosisrequires autocrine factors secreted by adjacent bacilli for the smallinocula to grow. Supplement of such factors from ESPSN or SPSN or cellextract containing the autocrine factors to the culture media canpotentially allow small inocula to grow, which otherwise cannot grow.Thus this method of cultivation using ESPSN, or SP SN, or cell extractof M. tuberculosis complex organisms can be used to improve thesensitivity of isolation of M. tuberculosis complex from clinicalspecimens, and also for improved growth of the M. tuberculosis complexorganisms for research lab use. Therefore, while the methodologydescribed herein utilizes M. tuberculosis in many examples, one of skillin the art will recognize that it can readily be adapted to othermycobacterial species.

[0027] By ESPSN we mean the supernatant obtained from the earlystationary phase of a culture, e.g. for M. tuberculosis complex asupernatant obtained from an approximately 3-4 week old culture. By SPSNwe mean the supernatant obtained from the stationary phase, e.g. for M.tuberculosis complex from an approximately 1-2 month old culture. By“isolated and sterilized” we mean that the supernatant has been treatedto remove particulate matter and sterilized to eliminate mycobacteria orother contaminating organisms. Collection of the supernatant, removal ofparticulate matter, and sterilization may be accomplished by any of avariety of means which are well known to those of skill in the art. Forexample, collection may be accomplished via centrifugation (e.g. about6,000× g for about 20 minutes) followed by sterilization via filtration,e.g. through a 0.22 μm filter. Those of skill in the art will recognizethat other suitable means of accomplishing collection and sterilizationof the supernatant are available and well-known, including but notlimited to centrifugation followed by UV irradiation.

[0028] As demonstrated herein, the isolated ESPSN and SPSN from M.tuberculosis complex contain factors which promote the revival orresuscitation of dormant mycobacterium bacilli. In order to be utilizedfor this purpose, the isolated ESPSN or SPSN may be used alone, or maybe supplemented or enriched with various nutrients in order to enhancegrowth of a new inoculum. Those of skill in the art will recognize thatmedia comprised of ESPSN or SPSN may be enriched by the addition ofother substances known to be conducive to the growth of bacteria ingeneral, and of mycobacterium in particular, for example, Tween 80,albumin-dextrose-catalase, various salts and nutrients, bufferingagents, fatty acids and the like. Nutrients may be added individually tothe ESPSN or SPSN . Alternatively, the ESPSN or SPSN may be combinedwith other fresh media to supply the nutrients.

[0029] In order to obtain ESPSN, SPSN or cell extracts, a culture ofmycobacterium must be established. Methods of culturing mycobacteriumare well-known to those of skill in the art (see, for example Kent, P T,Kubica, G P. Public health mycobacteriology. A guide for the level IIIlaboratory. Atlanta, Georgia; Centers for Disease Control, 1985; Nolte,F S, Metchcock B. Mycobacterium. In: Murray P R, Baron E J, Pfaller M A,Tenowver F C, Yolken R H ed. Manual of clinical microbiology, 6th ed.Washington D.C.; ASM Press 1995: pp 400-437) as are methods of obtaininga suitable supernatant from such a culture. In general, an inoculum froman appropriate strain of mycobacterium is introduced into a culturemedium and allowed to grow for the requisite period of time underconditions that are well-known to those of skill in the art (e.g.sterile conditions, about 37° C., with or without agitation of theculture). Suitable strains of mycobacterium which may be utilized in thepractice of the present invention include but are not limited to M.tuberculosis H37Ra, M. fortuitum, etc. Useful media for culturing M.tuberculosis strains include but are not limited to 7H9, 7H10, 7H11,7H12B, Sauton's medium, Dubos medium, egg-based media such asLowenstein-Jensen medium, and the like, which are readily commerciallyavailable (e.g. Difco). Further, those of skill in the art willrecognize that such media may be supplemented with substances such asTween 80 (0.05%), albumin-dextrose-catalase (ADC), fatty acids, and thelike.

[0030] The inoculated cultures are grown under suitable conditions untilearly stationary phase (ESP) is achieved. Typically, for M. tuberculosiscomplex the culture will be grown for approximately 3-4 weeks, and theoptical density of the culture at 600 nm will be in the range of about1.0 to 1.5, and more preferably will be in the range of about 1.0 to1.2. Alternatively, if SPSN is to be utilized, the culture is grown forabout 1-2 months prior to obtaining the supernatant. The supernatant canthen be isolated and sterilized as described above, and utilized torevive or resuscitate dormant bacilli.

[0031] By “revive or resuscitate dormant mycobacterium bacilli” and“enhance growth” we mean that a significant increase in the growth of asample containing dormant bacilli is observed when the sample iscultured in the media of the present invention, compared to the growthof an equivalent sample cultured in conventional media. An equivalentsample would be one in which an equal amount of inoculum was introducedinto an equal volume of media, and in which all other variables otherthan the presence/absence of isolated supernatant or components thereof,(e.g. temperature, degree of aeration, time of culturing, and the like)are held constant. By “increase in growth” we mean an increase in thetotal number of bacteria in the culture, as determined by any of severaltechniques that are well-known to those of skill in the art. Suchtechniques include ascertaining the number of colony forming units(CFUs) present in the culture after incubation for a fixed amount oftime, or by ascertaining the number of bacteria which test live in anFDA-EB test. In a preferred embodiment of the present invention, thequantitation of growth in the conventional and the supplemented mediummay be carried out after a suitable time. For example, the culturesshould be incubated for a minimum of about 2 days and for a maximum ofabout 7 to 28 days. The determination of growth in a culture may becarried out by any of a variety of techniques that are well-known tothose of skill in the art, including but not limited to plating on solidmedia and visually observing colony formation (i.e. determining CFUs),observing an increase in turbidity in liquid culture as measured byoptical density at absorption A600, or by utilizing a viability assaywith redox dye such as MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium), or by detectingan increase in growth index (GI) value due to production of C¹⁴-CO₂ inthe Bactec TB460 system, and the like. The increase in growth that isobserved, in order to be considered “significant” is preferably at leastabout a two fold increase in growth. Alternatively, what may be observedis the emergence of any growth at all compared to a control in which nogrowth is observed without the resuscitation factors. In yet anotherembodiment of the present invention, a media is provided which isprepared from ESPSN and/or SPSN combined with fresh, conventional media.Examples of suitable media include but are not limited to 7H9, 7H10,7H11, 7H12B, Sauton's medium, Dubos medium, egg-based media such asLowenstein-Jensen medium, and the like, which are readily commerciallyavailable (e.g. Difco). Further, those of skill in the art willrecognize that such media may be enriched with suitable substances suchas Tween 80, albumin-dextrose-catalase (ADC), fatty acids, and the like.The medium of the present invention may be prepared by combining ESPSNand/or SPSN with any suitable media, so long as the resulting mediumappropriately sustains the growth of mycobacterium and supports therevival or resuscitation of dormant bacilli. Generally, the ratio ofESPSN and/or SPSN to media will be in the range of from about 0.1:1 toabout 1:10, and more preferably in the range of from about 1:1 to about1:2. Further, the media may also be further supplemented by the additionof substantially purified components of ESPSN and/or SPSN such as theproteins, peptides and phopholipids (or mixtures thereof) describedbelow.

[0032] In another embodiment, the present invention further providesmedia for culturing mycobacterium which is comprised of a suitableculture medium supplemented with cell extract at least one substantiallypurified product of M. tuberculosis complex, the product exhibitinggrowth enhancing/resuscitation activity for dormant bacilli of themycobacterium. Such products may be obtained from, for example, theESPSN or the SPSN of a M. tuberculosis complex culture, or from cellularextracts of such a culture. While the examples recited herein discloseproducts from the ESPSN or the SPSN, those of skill in the art willrecognize that those products are of cellular origin and are releasedinto the supernatant from the bacteria. Thus, the products may also beobtained directly from cell extracts prior to their release from thecell. Alternatively, crude cell extracts may also be utilized. Methodsof obtaining such extracts are well known to those of skill in the artand include but are not limited to sonication, French press, grindingwith mortar and pestle, and the like. The amount of cell extract thatcan be added to fresh media to fabricate the supplemented media is inthe range of about from 1:10 to about 1:1000, cell extract: media.

[0033] By “substantially purified product” we mean a product which hasbeen purified to contain no more than about 0-20%, and more preferablyabout 0-10%, and even more preferably about 0-5% extraneous material.Those of skill in the art will recognize that substantially purifiedcomponents may contain trace amounts of material such as salts, ions(e.g. metal ions), and various other extraneous materials that do notinterfere with the resuscitation activity exhibited by the substantiallypurified components.

[0034] In a preferred embodiment of the present invention, themycobacterium is M. tuberculosis and the component of ESPSN and/or SPSNor cell extract is the protein Rv1147c or a peptide fragment of proteinRv1147c. By “protein Rv1147c” we mean the protein corresponding to theamino acid sequence encoded by the open reading frame of the M.tuberculosis genome sequence which has been designated M. tuberculosisRv1147c (accession number F70875).

[0035] Those of skill in the art will recognize that other amino acidsequences which are not absolutely identical to the sequence of proteinRv1147c may also be utilized in the practice of the present invention.For example, proteins with various amino acid substitutions, or withvarious deletions or insertions in the sequence (e.g. such as those thatmay occur in variants of M. tuberculosis or which are generated viagenetic engineering, etc.), or with various chemical modifications (e.g.acylation of the carboxy terminus), may also be utilized, so long asthey retain the ability to function in the practice of the presentinvention. In general, such Rv1147c-based proteins will possess highhomology to Rv1147c, i.e. in the range of about 75 to 100% homology, ormore preferably in the range of about 85 to 100% homology, and mostpreferably in the range of about 95-100% homology to Rv1147c. Further,the protein Rv1147c may be from any source (e.g. isolated from M.tuberculosis, or from another organism into which the gene encoding theprotein has been cloned, or fabricated synthetically, etc.)

[0036] Further, the component of ESPSN and/or SPSN or cell extract maybe a peptide fragment of Rv1147c. Examples of such peptide fragmentsinclude but are not limited to those with the amino acid sequences ofSEQ IDS NOS.1-5 of the instant invention. However, those of skill in theart will recognize that many variations of these peptides may be made(for example, by varying the primary sequence of the peptides by aminoacid substitutions, deletions or insertions, or by extending orshortening their length, etc.) and all such modified peptide fragmentsof Rv1147c are intended to be encompassed in the practice of the presentinvention. Any peptide fragment of Rv1147c may be utilized in thepractice of the present invention, including peptides which are based onor are obvious variants of SEQ IDS 1-5 . In general, the length of sucha peptide fragment of Rv1147c will be from about 5 to about 20 aminoacids. Further, the peptide fragments may be from any suitable source,e.g. they may be generated by chemical or proteolytic cleavage ofRv1147c or related proteins, they may be produced synthetically, or theymay be produced via genetic engineering techniques. The particularsource of the ESPSN and SPSN-based protein or peptides of the presentinvention is not a crucial feature of the invention.

[0037] The concentration of protein or peptide to be present in themedia of the present invention may vary depending on the resuscitationactivity of a given protein or peptide. However, it will generally be inabout the picomolar to micromolar range. Typically, the concentrationshould be adequate to provide a significant increase in the CFU formingability of dormant bacilli, compared to conventional, unsupplementedmedia.

[0038] In another embodiment of the present invention, the substantiallypurified component of ESPSN and/or SPSN or cell extract is aphospholipid or a component of a phospholipid. Phospholipids (alsocalled phosphoglycerides) are composed of glycerol, phosphate and twofatty acyl units. Thus fatty acid components can have a carbon chainlength of, for example, C18 (octadecanoic acid), C19 (nonadecanoicacid), 20 (eicosanoic acid), 21 (heneicosanoic acid), 22(docosanoicacid), 23 (tricosanoic acid) 24 ((tetracosannoic acid), 25(pentacosanoic acid), 26 (hexacosanoic acid), 27 (heptacosanoic acid),28 (octacosanoic acid), 29 (nonacosanoic acid), 30 (triacontanoic acid),31 (hentriacontanoic acid). Derivatives of these fatty acids, such aswith double bonds and esters of these fatty acids can, either alone orin combination, be added to the culture media (7H12B, 7H9, 7H10, 7H11,Sauton's medium, Dubos medium and egg-based media such asLowenstein-Jensen medium) for improved diagnosis and treatment ofmycobacterial infections. In a preferred embodiment of the presentinvention, the phospholipid or component of a phospholipid isphosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine,phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid,arachidonic acid, C18-C31 fatty acids with or without double bonds, andesters of C18-C31 fatty acids with or without double bonds. Suchphospholipids are well-known to those of skill in the art and arereadily available.

[0039] The concentration of phospholipid or component thereof to bepresent in the media of the present invention may vary depending on theresuscitation activity of a given phospholipid. However, it willgenerally be in about the picomolar to micromolar range. Typically, theconcentration should be adequate to provide a significant increase inthe CFU forming ability of dormant bacilli, compared to conventional,unsupplemented media.

[0040] In addition, the media of the present invention may comprise morethan one substantially purified component of ESPSN and/or SPSN or cellextract. For example, a combination of several peptide fragments, or ofseveral lipids, or of several peptides and several lipids may also beutilized. In this case, the concentration of each component willtypically be in the picomolar to micromolar range.

[0041] Types of media that may be supplemented by components of ESPSNand/or SPSN or cell extract include but are not limited to 7H12B, 7H9,7H10, 7H11, Sauton's medium, Dubos medium and egg-based media such asLowenstein-Jensen medium. Further, those of skill in the art willrecognize that the media of the present invention may be provided in anyof several suitable forms. For example, the media may be provided in apremixed form (i.e. the components have already been added) or thecomponents may be provided separately for addition to conventionalmedia. Further, the media may be liquid (ready to use or concentrated)or solid.

[0042] The invention further provides methods for reviving dormantbacilli of a mycobacterium species. In one embodiment of the presentinvention, the method involves exposing dormant bacilli to isolatedESPSN or SPSN from M. tuberculosis complex cultures. The isolated ESPSNand SPSN will preferably have been sterilized, e.g. by filtersterilization.

[0043] In another embodiment of the present invention, the revivalmethod involves exposing dormant bacilli of a mycobacterium species to acell extract or at least one substantially purified product of M.tuberculosis complex (e.g. from the ESPSN, the SPSN, or a cell extract),the product exhibiting resuscitation activity for dormant bacilli ofmycobacterium. In one embodiment, the product is a phospholipid orcomponent thereof. In a preferred embodiment of the present invention,the phospholipid is phosphotidyl-L-serine, dioleoylphosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine,tuberculostearic acid, arachidonic acid, or a fatty acid. In anotherembodiment, the product is a protein. In a preferred embodiment of thepresent invention the protein is Rv1147c or a peptide fragment ofprotein Rv1147c. Exemplary peptide fragments of Rv1147c include thepeptides corresponding to SEQ ID NOS. 1-5.

[0044] Methods for the diagnosis of infections caused by a mycobacteriumspecies (such as Mycobacterium tuberculosis, Mycobacteriumparatuberculosis, and Mycobacterium leprae) are also contemplated.Improved methods are needed because, in the case of, for example,tuberculosis (caused by an M. tuberculosis infection), clinicalspecimens from suspected TB patients, especially those from patientsunder treatment, are known to contain injured and dormant bacilli, whichmay not grow in conventional culture media. In addition, bacilli in thespecimens will also suffer from significant stress and injury during theprocessing of the clinical specimens by strong alkali NaOH and thecentrifugation heat. Addition of the growth enhancing/resuscitationfactors is likely to resuscitate the dormant or injured bacilli in theclinical specimens, resulting in a better recovery rate or improvedsensitivity of primary isolation or the bacilli. The diagnostic methodsof the instant invention are geared to detecting live and/or dormant M.tuberculosis bacilli with a far greater level of confidence. Thediagnostic methods of the present invention may be used, for example, toscreen persons suspected of having tuberculosis, or to monitor theprogress of eradication of M. tuberculosis bacilli during or aftertreatment. In whatever context the diagnostic method is used, it wouldbe highly advantageous to have the ability to detect dormant or injuredforms of bacilli.

[0045] In one embodiment, the diagnostic method involves combining asample for which the presence or absence of a mycobacterium species isto be ascertained with isolated early stationary phase supernatant of M.tuberculosis complex. The isolated ESPSN may have been sterilized e.g.by filter sterilization.

[0046] In another embodiment of the present invention, the diagnosticmethod involves combining a sample for which the presence or absence ofa mycobacterium species is to be ascertained with a cell extract or atleast one substantially purified product of M. tuberculosis complex(e.g. a component of ESPSN and/or SPSN culture supernatant or of a cellextract of the mycobacterium species). The cell extract or productexhibit the property of enhancing the growth of or resuscitating dormantbacilli of mycobacteria. In one embodiment, the product is aphospholipid or component of a phospholipid. In a preferred embodiment,the phospholipid or component thereof is, for example,phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine,phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid,arachidonic acid, or a fatty acid. In another embodiment, the product isthe protein Rv1147c or a peptide fragment of protein Rv1147c. Exemplarypeptide fragments of Rv1147c include the peptides corresponding to SEQID NOS. 1-5. Further, the method may also utilize combinations of suchproducts.

[0047] Suitable samples for analysis by the methods of the presentinvention include but are not limited to sputum, blood, tissue, and thelike.

[0048] The present invention also provides a kit for use in thediagnosis of infections caused by a mycobacterium species (such asMycobacterium tuberculosis, Mycobacterium paratuberculosis, andMycobacterium leprae. In one embodiment, the kit comprises a sealedcontainer comprising isolated ESPSN and/or SPSN of M. tuberculosiscomplex. The isolated supernatant may have been sterilized e.g. byfilter sterilization. In another embodiment of the invention, the kitcomprises a sealed container comprising cell extract or at least onesubstantially purified product of M. tuberculosis complex, the productdisplaying the property of resuscitating dormant bacilli ofmycobacteria, or generally enhancing the growth of the mycobacteria. Inpreferred embodiments, the product is a component of ESPSN, SPSN, or acell extract of M. tuberculosis complex. In one embodiment, thecomponent is a phospholipid or component of a phospholipid. In apreferred embodiment, the phospholipid or component thereof is, forexample, phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine,phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid,arachidonic acid, or a fatty acid. In another embodiment, the product isa protein. In a preferred embodiment of the present invention, theprotein Rv1147c or a peptide fragment of protein Rv1147c. Exemplarypeptide fragments of Rv1147c include the peptides corresponding to SEQID NOS. 1-5. Further, the kit may also comprise combinations of suchproducts, as well as such items as instructions for use of the kit.

[0049] Another aspect of the present invention is the treatment ofinfections caused by a mycobacterium species (such as Mycobacteriumtuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae).The treatment method involves the administration to a patient cellextract or at least one substantially purified product of M.tuberculosis complex, the product or extract displaying the property ofresuscitating dormant bacilli of mycobacteria, or generally enhancingthe growth of mycobacteria. In preferred embodiments, the product is acomponent of ESPSN, SPSN, or a cell extract of M. tuberculosis complex.In one embodiment, the component is a phospholipid or component of aphospholipid.

[0050] In a preferred embodiment, the phospholipid or component is, forexample, phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine,phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid,arachidonic acid, or a fatty acid. In another embodiment, the product isa protein. In a preferred embodiment of the present invention, theprotein is Rv1147c or a peptide fragment of protein Rv1147c. Exemplarypeptide fragments of Rv1147c include the peptides corresponding to SEQID NOS. 1-5. The components is administered in conjunction with anestablished drug treatment regimen such as DOTS. The administration ofsuch components serves to revive dormant bacilli in the patient, makingthem susceptible to eradication by drug therapy. Otherwise, onlynon-dormant bacilli are eliminated and the patient is at risk forrelapsing due to revival of the dormant bacilli after the treatmentregimen has ceased. A combination of the above (e.g. more than onephospholipid, more than one peptide, or a combination of lipids andpeptides) may also be administered. The quantity of the component to beadministered may vary from patient to patient, depending on factors suchas weight, gender, age, and general health, etc. of the patient, and itbest determined by a skilled practitioner such as a physician.Generally, however, when a phospholipid is administered, the dosage willbe in about the micromolar range, and when a protein or peptide isadministered, the dosage will be in about the micromolar range.

[0051] Administration may be effected by any of a variety of routes thatare well-known to those of skill in the art, including but not limitedto oral, perenteral, intravenously, via inhalation, and the like.

[0052] To that end, the invention also provides a pharmacologicalpreparation comprising cell extract or at least one substantiallypurified product of M. tuberculosis complex, the product displaying theproperty of resuscitating dormant bacilli of mycobacteria, or generallyenhancing the growth of mycobacteriu. The component may be aphospholipid or a component of a phospholipid, or a protein or fragmentof the protein. In a preferred embodiment of the present invention, thephospholipid or component of a phospholipid is, for example,phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine,phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid,arachidonic acid, or a fatty acid. In another embodiment, the componentis the protein Rv1147c or a peptide fragment of protein Rv1147c.Exemplary peptide fragments of Rv1147c include the peptidescorresponding to SEQ ID NOS. 1-5. The pharmalogical preparation may alsocomprise a combination of the above (e.g. more than one phospholipid,more than one peptide, or a combination of lipids and peptides. Such apharmacological preparation may also comprise other suitable substancesand excipients, including but not limited to physiological acceptablebuffering agents, stabilizers (e.g. antioxidants), flavoring agents, andthe like. Further, the pharmacological preparation may be in any of avariety of art-accepted forms such as tablets, capsules, variousinjectable formulations, and the like, as are suitable for the desiredmeans of administration.

[0053] In yet another aspect, the present invention provides a method ofinhibiting the growth of a mycobacterium species such as Mycobacteriumtuberculosis, Mycobacterium paratuberculosis, or Mycobacterium leprae.The method involves exposing the mycobacterium species to the isolatedand sterilized supernatant of a culture M. tuberculosis complex or acell extract of M. tuberculosis complex that is at least about 3 monthsin age. Alternatively, the method involves exposure of the mycobacteriumspecies to substantially purified products from such a culture, whereinthe products exhibit the property of inhibiting the growth of themycobacterium species.

EXAMPLES Methods

[0054] Bacterial Culture Conditions.

[0055]M. tuberculosis H37Ra cultures were grown in 7H9 medium containingTween 80 (0.05%) and ADC (albumin-dextrose-catalase) enrichment at 37°C. for various periods of time as standing batch cultures. Cultures ofvarying age up to one year were used for the resuscitation experiments.Resuscitation of dormant M. tuberculosis by phophatidylserine wasperformed as follows. A 6 month old M. tuberculosis H37Ra culture waswashed and resuspended in 7H9 medium followed by plating at differentdilutions on 7H11 agar plates containing 4 μg/ml phosphatidylserine(derived from bovine brain, containing a mixture of two unknown fattyacyl groups, Sigma Chemical Co.) or phosphatidylserine dioleoyl. Theplates were incubated at 37° C. for 3 weeks when the CFU was determined.Data represent average of duplicate samples.

[0056] Purification of Resuscitation Activity.

[0057] Spent culture supernatant from a 4-week-old stationary phase M.tuberculosis H37Ra culture grown in Sauton's medium (4 liters) wascollected by centrifugation at 8000 rpm for 15 minutes. The supernatantwas then passed through a SepPak Aluminum N column (Waters, Inc), whichwas washed with hexane and eluted with methanol:chloroform (1:2). Theeluted materials were dried with Rotavapor R-3000 (BUCHI) and the driedmaterials were dissolved in a small volume of methanol:chloroform (1:2)and further fractionated by preparative thin layer chromatography onsilica gel G60 using solvent system (chloroform:acetone:methanol:aceticacid:water=10:4:2:2:1). The silica gel from TLC plate was divided into10 fractions, which were eluted with methanol:chloroform (1:2), dried bySpeedVac and assayed for resuscitation activity on the 6 month old H37Rabacilli by FDA-EB staining. Active TLC fractions were pooled and furtherfractionated by reverse phase high-performance liquid chromatography(HPLC) on a Spectraphysics instrument, using a narrow bore mixed bed C18cation-exchange column (Alltech), and a gradient using the followingbuffers: Buffer A (0.1% TFA in water) and Buffer B (0.1% TFA inacetonitrile). The eluate was monitored at 214 nm and 280 nm. Sixty HPLCfractions were collected and assayed for resuscitation activity on a 6month old H37Ra culture using fluoroscein diacetate (FDA)-ethidiumbromide (EB) viability staining (4), where live cells stained green anddead or injured cells red.

[0058] Mass Spectroscopic Analysis.

[0059] The active fractions were analyzed by matrix assisted laserdesorption/ionization (MALDI) using a Kompac 4 MALDI instrument fromKratos. Fractions were analyzed in both positive and negative ion mode,using an extraction voltage of 20 kV and a 337 nm nitrogen laser. Allspectra were the average of 50 laser shots. The matrix used was asaturated solution of α-cyano-4-hydroxycinnamic acid in 50% ethanol.

[0060] Protein Sequencing and Peptide Synthesis.

[0061] Protein sequencing of fractions 43 and 44 was performed in theProcise T Protein Sequencing System machine (Applied Biosystems) byEdman degradation method, at Synthesis & Sequencing Facility of JohnsHopkins University School of Medicine. Overlapping synthetic peptidesthat cover the 8,331 Dalton secreted polypeptide:

[0062] P1 (NH2-DPVDAVINTTCNYGQVVAALNATD-OH) (residues 29-52; SEQ ID NO.1);

[0063] P2 (NH2-LNATDPGAAAQFNASPVAQSYLRN-OH) (residues 48-71; SEQ ID NO.2);

[0064] P3 (NH2-LRNFLAAPPPQRAAMAAQLQAV-OH) (residues 69-90; SEQ ID NO.3); and

[0065] P4 (NH2-AQLQAVPGAAQYIGLVESVAGSCNNY-OH) (residues 85-110; SEQ IDNO. 4);

[0066] were synthesized at Synthesis & Sequencing Facility of JohnsHopkins University School of Medicine. Synthesis of P1 peptide wasunsuccessful due to extreme hydrophobicity. The synthetic peptides werepurified by HPLC and were over 95% pure.

Example 1 The Resuscitation Phenomenon in M. tuberculosis

[0067]M. tuberculosis H37Ra was cultivated in 7H9 Tween 80 (0.05%)albumin-dextrose-catalase liquid medium (Difco) for various times,ranging from a few weeks to several months and sometimes up to 2-3 yearsas standing batch cultures at 37° C. with occasional agitation. Thestanding aged batch cultures settled to the bottom of culture tubes withno apparent surface growth. The early stationary phase culturesupernatant (ESPSN) from a 3-4 week old M. tuberculosis H37Ra standingculture grown in 7H9 medium was collected following centrifugation at6,000 g and sterilized by filtration through 0.22 μm filter. Thesterilized ESPSN did not form colonies on 7H11 agar plate. Portions ofbacterial cell pellets (about 10⁷⁻⁸ bacilli in 100 μl) prepared from an8 month old standing H37Ra batch culture were resuspended in the samevolume (100 μl) of its own aged culture supernatant (A), fresh 7H9medium (B), and filtered ESPSN (C). Upon incubation at 37° C. for 3 dayswithout shaking, CFU in A, B, and C was determined by platingappropriate dilutions of the cell suspensions on 7H11 agar platesfollowed by incubation for 4 weeks at 37° C. The data are presented inTable 1. The bacilli from the 8 month old aged culture gave almost 1000fold less colonies in its own aged culture supernatant than in freshmedium. This suggests that aged culture supernatant contained growthinhibitory activity. On the other hand, the aged bacilli incubated inESPSN produced about 20 fold more colonies than the fresh medium control(Table 1). This indicates that ESPSN allowed a population ofnonculturable bacilli (injured or dormant bacilli) to form colonieswhich otherwise failed to do so in fresh medium. The above phenomenonhas been reproduced many times with different batches of aged M.tuberculosis H37Ra cultures of varying age up to 2-3 years using thesame ESPSN. The resuscitation or growth stimulation phenomenon was alsofound with the bacilli grown in Sauton's medium, indicating that thetype of medium is not important for production of the resuscitationactivity by tubercle bacilli. TABLE 1 Resuscitation activity in theearly phase culture supernatant 8 month old tubercle bacilli* Treatmentin various media plating efficiency (cfu/ml) (A) own culture media 2.0 ×10³ (B) 7H9 medium control 1.2 × 10⁶ (C) ESPSN 2.4 × 10⁷

Example 2 Monitoring the Resuscitation Phenomenon by FluorosceinDiacetate-Ethidium Bromide (FDA-EB) Staining

[0068] Because cfu determination is time-consuming (4-6 weeks), we usedFDA-EB staining (Kvach and Veras, 1982) to more rapidly assess theviability status of aged M. tuberculosis cultures upon treatment withESPSN. The principle of FDA-EB staining is as follows: FDA crosses themembranes of dead and live cells, hydrolyzed into free fluorescein byboth types of cell, but the latter is retained only by live cells withintact membrane. On the other hand, EB only enters dead cells or cellswith impaired membrane integrity and stains DNA. Green cells stained byFDA were considered live cells, orange-red cells stained by EB deadcells, and dual-stained cells injured or dormant cells (Kvach and Veras,1982). Briefly, mycobacterial cultures (100 μl) were stained for about20 minutes with 50 μl FDA-EB working solution containing FDA and EB at 2μg/ml and 4 μg/ml diluted from stock solution of FDA (5 mg/ml inacetone) and EB (2 mg/ml in PBS) in PBS buffer. The stained mycobacteriawere examined under a fluorescence microscope with FITC (fluoresceinisothiocyanates) filter with excitation at 490 nm and emission at 525nm. FDA-EB staining has been shown to correlate with the viability orcfu of mycobacteria (Kvach and Veras, 1982). Using the FDA/EB staining,we examined the resuscitation phenomenon by ESPSN on bacilli from agedH37Ra standing batch cultures grown in 7H9 medium of varying age rangingfrom 8-16 months. Bacilli (about 10⁸⁻⁹ cells/ml) from various agedcultures were incubated with ESPSN or control 7H9 medium in a volume of100 μl at 37° C. for 2 days followed by FDA-EB staining and fluorescencemicroscopy. The viability of the bacilli was determined by calculatingthe average percentage of green cells over total number of cellsobserved for at least 5 views under the microscope. Five to 20 fold moregreen cells were found when the bacilli from various aged cultures weretreated with ESPSN than with fresh medium control (Table 2). Theseresults suggest that the bacilli became more viable after resuscitationwith ESPSN and that FDA/EB staining is a quick way to monitor theresuscitation process. Culture supernatants from early stationary phasecultures of M. smegmatis or E. coli had no effect on resuscitation orgrowth stimulation of aged tubercle bacilli. TABLE 2 Effect ofresuscitation medium (ESPSN) on the resuscitation of M. tuberculosiscultures of varying age by FDA-EB staining Percentage (%) of green(live) cells determined by FDA-EB staining Culture Age Direct stainingFresh 7H9 medium control ESPSN  8 month 1.6 9.4 49.8 10 month 1.8 2.449.2 15 month 0 (all orange-red) 0.8 19.4 16 month 0 (all orange-red) 0(all orange-red) 11.2

Example 3 Expression and Localization of the Resuscitation Activity.

[0069] To determine the presence of resuscitation activity in relationto growth phase, portions of filter-sterilized culture supernatant (100μl) taken at different growth stage of an H37Ra standing culture (up to8 weeks at 37° C.) were assayed on about 10⁷⁻⁸ bacilli from the same 8month old culture as described in Table 1 using the FDA-EB staining. Theresuscitation activity was present mainly from early stationary phase(3-4 weeks old) onwards up to 2 months we examined. There was hardly anyresuscitation activity in the log phase (1-2 weeks old) culturesupernatant (data not shown). To determine where the resuscitationactivity is located, the culture supernatant and bacterial lysate of a3-4 week old M. tuberculosis H37Ra culture were prepared and assayed forresuscitation activity similarly. The lysate was reconstituted to theoriginal volume of the culture using 7H9 medium and sterilized byfiltration through a 0.22 μm filter before use. The lysate was found tohave about 1/40th of the activity as compared with that in thesupernatant, indicating that the resuscitation activity is mainlypresent in the culture supernatant and only a small amount of thisactivity is present in the cell.

Example 4 The ESPSN Allowed Smaller Bacterial Inocula to Start CultureGrowth than Fresh Medium

[0070] To start an M. tuberculosis culture requires a relatively largeinoculum, and a small inoculum often fails to initiate the growth of M.tuberculosis in liquid culture (Dubos and Davis, 1946). The reasonbehind this observation is unknown. We tested if the ESPSN couldinfluence the size of bacterial inoculum required to initiate growth oftubercle bacilli in liquid culture. To do this, a 6 month old M.tuberculosis H37Ra batch culture that had been kept at 37° C. withoutshaking was 10 fold serially diluted (0.4 ml into 3.6 ml) intofilter-sterilized ESPSN (prepared from a 4 week old H37Ra standingculture grown in Sauton's medium) and into a control Sauton's medium.The Sauton's medium contained detergent Triton WR1 339 at 0.025% toreduce bacterial clumping. The various dilutions were incubated at 37°C. for 2 weeks when the visibility of bacterial growth and cfu weredetermined. ESPSN allowed smaller inocula (10⁻³ to 10⁻⁴) to form visiblegrowth in liquid subcultures which otherwise failed to give any visiblegrowth in Sauton's medium alone (Table 3, Panel A). The growthstimulating or resuscitation effect was also reflected in the increasedcfu over the medium control (Table 3, Panel B). Strikingly, no cfu wasdemonstrable for 10⁻³ to 10⁻⁵ dilutions in medium control when directlyplated, whereas 10⁴ to 10⁵ cfu/ml were detected for the same dilutionsin ESPSN. Neither ESPSN alone nor the 10-6 dilutions in ESPSN or freshmedium gave any bacterial growth in liquid culture or on agar plates.

[0071] The age of the culture where the spent culture supernatant isderived is important. Log phase culture supernatant of MTB (less than 2week old) has little growth enhancing or resuscitation activity. Earlystationary and stationary phase culture supernatant (from 3-4 weeks upto 2 months old) have significant growth enhancing or resuscitationactivity for MTB. Old spent culture supernatant of MTB (e.g., 3-12months old culture) has growth inhibitory effect or toxic effect on thegrowth of MTB. TABLE 3 Growth stimulation of 6-month-old M. tuberculosiscultures by ESPSN with small bacterial inocula Visible growth^(a)CFU/ml^(b) Dilution Sauton's ESPSN Sauton's ESPSN 10⁻¹ + + 5 × 10⁶ >10⁷10⁻² + + 10⁶   10⁷ 10⁻³ − + ND 3 × 10⁵ 10⁻⁴ − + ND 2 × 10⁵ 10⁻⁵ − − ND 3× 10⁴ 10⁻⁶ − − ND ND

Example 5 Characterization of the Resuscitation Activity

[0072] To determine the properties of the resuscitation activity inresponse to physicochemical factors as well as the nature of activity,we subjected the ESPSN to various treatments followed by assaying theresuscitation activity using the FDA-EB viability staining andfluorescence microscopy. In all the following experiments, the same 8month old culture as described in Table 1 and a positive control ESPSNwere used.

[0073] (a) Effect of temperature: Heat treatment of ESPSN at 100° C. for10 minutes had no effect on the resuscitation activity, indicating thisresuscitation factor is heat stable. This experiments also rules out thepossibility that any residual bacilli or filterable form of the bacilliin the filtered culture supernatant may have contributed to theincreased number of bacilli upon resuscitation. In addition, repeatedfreeze (−70° C.)/thaw (37° C.) for 10-15 cycles and storage of the ESPSNat -70° C. for 12 months had no significant effect on the resuscitationactivity (data not shown).

[0074] (b) Effect of acid and alkali. ESPSN was incubated with 1 N HClor 1 N NaOH for 1 hour at room temperature followed by neutralizationwith the same molar concentration of alkali or acid and was tested forresuscitation activity. Acid treatment completely abolished theresuscitation activity and alkali also inhibited the activity but to alesser extent.

[0075] (c) Effect of pH on resuscitation phenomenon. To determine the pHconditions that may affect the resuscitation phenomenon, we adjusted thepH of ESPSN to 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 and sterilized themedium by filtering through a 0.22 μm filter. The cell pellets from an18 month old H37Ra culture (100 111) were resuspended in the ESP SNadjusted with various pH values followed by incubation at 37° C. for 2days and the effect of resuscitation was examined by FDA-EB staining.Fresh 7H9 liquid medium was also adjusted to the above same pH valuesand included as controls. The resuscitation occurred mainly at neutralpH but not at acid pH.

[0076] (d) Nature of the resuscitation activity. RNase T1, DNase I,Exonuclease III were added to ESPSN at 2000 u/ml, 100 u/ml, 150 u/ml,respectively. Following incubation at 37° C. for 90 minutes, the enzymeswere inactivated by heating at 70° C. for 15 minutes. Proteinase K,pronase, and trypsin were added to ESPSN at 20 μg/ml, 250 μg/ml and 25μg/ml, respectively, and the mixtures were incubated at 37° C. for 1hour followed by heat inactivation at 100° C. for 10-15 minutes. Thesetreatments did not affect the resuscitation activity, indicating thatthe resuscitation factor is unlikely to be a polypeptide or nucleicacid.

[0077] (e) Preliminary determination of the molecular weight of theresuscitation activity. Bio-Gel-P2 (Bio-Rad) which separates compoundswith small molecular weight (exclusion limit 1,800 Dalton) was used.Before the ESPSN sample was loaded, a molecular weight standard(Thyroglobulin, 670 kD; Bovine gamma globulin, 158 kD, Chickenovalbumin, 44 kD; Equine myoglobin, 17 kD; and Vitamin B 12, 1.375 kD)was run and a standard curve was established with standard molecularweight versus elution volumes. After the ESPSN sample was loaded, thecolumn was eluted with 7H9 medium without ADC, and various fractionswere filtered through a 0.22 μ filter before being tested forresuscitation activity using FDA/EB staining. The resuscitation activitywas found to be smaller than the Vitamin B12 standard, which is 1,375Dalton. Consistent with this finding, we found that dialysis of ESPSNusing dialysis tubing with molecular weight cutoff of 3,000 Daltoncaused complete loss of the activity (data not shown).

Example 6 Identification of Phospholipids as Having ResuscitationActivity from Culture Supernatants

[0078] To identify the active components responsible for theresuscitation activity, spent culture supernatant from a stationaryphase M. tuberculosis H37Ra culture was subjected to concentration andfractionation. Sixty HPLC fractions were assayed for resuscitationactivity on a 6-month-old H37Ra culture using fluoroceindiacetate-ethidium bromide (FDA-EB) viability staining. Fractions 1-4,and 46 had killing activity as judged by more EB-stainable red cellsthan control. Fractions 8, 22 and 23 and fractions 43 and 44 hadprominent resuscitation activity as judged by presence of moreFDA-stainable green cells. Analysis of fraction 8 was unsuccessful.Matrix-assisted laser desorption/ionization (MALDI) mass spectraobtained for fractions 22 and 23 both had a major peak at m/z 782 and asecond peak at m/z 621. Presuming that the peak at m/z 782 is theprotonated molecular ion and the second peak a fragment, the differenceof 141 mass units is characteristic of the loss of a phosphoethanolaminehead group from a phosphatidylethanolamine molecule (Heller et al.,1988) with an isotopically averaged molecular mass of 781. The remainingmass of the diacyl glycerol moiety may be accounted for by two fattyacids comprised of 39 carbons and 4 double bonds (C39:4). A compositionof a C19:0 (RI) and C20:4 (R2) would be consistent with the molecularand fragment masses observed, and could include tuberculostearic acid[CH₃(CH₂)₇CH(CH₃)—(CH₂)₈COOH] and arachidonic acid[CH₃(CH₂)₃(CH₂CH═CH)₄—(CH₂)₃COOH].

[0079] To confirm that a phospholipid was responsible for activity, wefirst tested commercially available phosphatidyl-L-serine and a dioleoylphosphatidyl-L-serine, both of which are precursor ofphosphatidylethanolamine and phosphatidylcholine, for resuscitationactivity for the 6 month old M. tuberculosis H37Ra cells. Both compoundshad significant resuscitation activity over medium control as judged bycolony forming units (CFU) assay. Phosphatidyl-L-serine and a dioleoylphosphatidyl-L-serine at 4 μg/ml gave 1.2×10⁷, 2×10⁶ CFU/ml,respectively, whereas the medium control produced 3×10⁴ CFU/ml.Phospholipase A2 and phospholipase C abolished the resuscitation ofthese compounds, whereas phospholipase D did not affect the growthenhancing effect of the phosphatidylserine derivatives (data not shown).In addition, phosphatidylcholine and phosphatidylethanolamine also hadsimilar resuscitation and growth enhancing activity for M. tuberculosisas phosphatidylserine or phosphatidyl-serine-dioleoyl. However,structural analog phosphatidylglycerol-oleoyl-palmitoyl did not havesignificant activity.

[0080] It is well known that to initiate growth of M. tuberculosis inliquid culture a large inoculum is required (Dubos and Davis, 1946). Ina separate experiment involving a fresh 4-week-old M. tuberculosis H37Raculture, the culture was serially diluted into Sauton's medium (1 ml)alone or into medium containing 5 μg/ml phosphatidylserine at 10⁻²,10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷ dilutions. The cultures were incubated at37° C. for 20 days when the visible growth was assessed and the cultureswere plated on 7H11 plates, which were incubated at 37° C. for 3 weeks.Phosphatidyl-L-serine allowed small inocula (10⁻⁷ dilution) to formvisible growth and CFU on plates, whereas the control culture grew onlyat 10⁻⁵ dilution. Taken together, these data suggest that thephospholipids not only resuscitated old tubercle bacilli but alsoallowed small inocula to initiate growth in liquid culture.

Example 7 Identification of an 8 kD Protein (Rv1174c) as HavingResuscitation Activity

[0081] MALDI mass spectroscopic analysis of fractions 43 and 44 bothrevealed a single peak at m/z 8,332, corresponding to the protonatedmolecular ion of a peptide with a molecular mass of 8,331 Daltons havingthe resuscitation activity. N-terminal amino acid sequencing identifiedthe following peptide sequence:

[0082] NH2-DPVDAVINTTCNYGQVVAALNATDPGAAAQ-OH (SEQ ID NO. 5).

[0083] Homology search revealed that the peptide was identical to aminoacid residues 29-58 of a hypothetical protein Rv1147c with unknownfunction from the M. tuberculosis genome sequence database (Cole et al.,1998). The predicted molecular mass of Rv1174c is 10,881 Dalton,suggesting that the first 28 amino acids of Rv1174c represents a signalsequence that is removed when secreted into the culture supernatant,giving rise to the 8,331 Dalton peptide.

[0084] To confirm that the Rv1174c has resuscitation activity, weattempted to overexpress this protein in E. coli. However, therecombinant Rv1174c was poorly expressed in E. coli. Nevertheless,culture supernatant of the recombinant E. coli strain expressing Rv1174c had resuscitation activity compared with vector control (data notshown). To circumvent this problem and bearing in mind that proteinsignaling molecules are often subject to proteolytic cleavage foractivity (Dunny and Leonard, 1997), we made synthetic peptides thatcover the 8,331 Dalton polypeptide and assayed their growth enhancingand resuscitation activity. Peptides P2, P3, P4 were tested on bothfresh M. tuberculosis cells (4 week old) and old cells of varying age upto one year. Significant growth enhancement or resuscitation activitywas observed for both types of cells, especially for old cells. PeptidesP2, P3, P4 had significant growth enhancing effect over the control on a4 week old H37Ra culture as judged by CFU counts (Table 4). The growthenhancing effect was more apparent after 5-day incubation with thepeptides. Peptide P4 appeared to be more active than P2 and P3 in growthstimulation. The growth enhancement effect of a mixture of the P2, P3,P4 was better than the individual peptides used singly (Table 4). TABLE4 Effect of Rv1174 peptides on growth of fresh M. tuberculosis cells(CFU values × 10⁵/ml) Days P2 P3 P4 P2 + P3 + P4 ESPSN 7H9 3 2.25 4.5 2015 2.25 1.5 5 5 450 32.5 75 100 6.5 8 150 1500 750 2500 150 10 11 7501500 2000 4250 750 35

[0085] The resuscitation activity of the peptides on a standing 7 monthsold H37Ra culture was then tested. The peptides produced 10 to over 100fold more CFUs than the control without peptides, and again the mixtureof the 3 peptides produced best result (Table 5, A). Results of MTTredox dye (10) and FDA-EB viability staining (Table 5, B and C)correlated with the CFU data. TABLE 5 Effect of Rv1174 peptides ongrowth of a 7-month-old M. tuberculosis H37Ra Days P2 P3 P4 P2 + P3 + P47H9 A. CFU data (× 10⁵ CFU/ml) 5 100 40 50 100 13 10 200 800 900 2750 20B. MTT data (OD readings at 570 nm) 0 0.2609 0.3147 0.2248 0.2365 0.24855 0.6269 0.6219 0.6287 0.6049 0.4803 10 1.1567 1.1917 1.4131 1.639710.774 C. FDA-EB viability staining (% of viable green cells) 0 1 2 1 1 25 2 4.5 11.5 10 2 10 27.5 42.5 50 67.5 12.5

[0086] This finding was surprising in view of the previous observation(see Example 5) that the resuscitation activity of the spent supernatantwas unlikely to be a polypeptide, in view of its insensitivity totreatment with Proteinase K, pronase, and trypsin.

Example 9 Antibody Raised Against the 8 kd-Derived Peptides Neutralizethe Resuscitation Activity

[0087] The ability of antibody raised against the peptides to block thegrowth enhancing effect of the peptides was assessed. Indeed, rabbitpolyclonal antiserum against P2, P3, P4 was found to antagonize thegrowth enhancement activity of the peptides, whereas pre-immunizationcontrol serum did not have this effect (Table 6). TABLE 6 Blocking ofgrowth enhancing effect of Rv1174 peptides by specific polyclonalantiserum. CFU data(CFU: × 10⁵/ml) P2 + P2 + P3 + P3 + P4 + P4 + P2 +P3 + P4 + P2 + P3 + P4 + 7H9 + pre Ab pre Ab pre Ab pre Ab pre 7H9 102.535 65 32.5 192.5 85 210.5 145.5 62.5 45

Example 10 Resuscitation of Truly Dormant Tubercle Bacilli that Did NotContain any CFU on Plates

[0088] The most dramatic effect of the peptides was seen on theresuscitation of dormant bacilli from a one-year-old culture that hadbeen incubated at 37° C. and dried up due to long term incubation andevaporation. The old culture when incubated in 7H9 liquid medium or 7H9medium plus rabbit serum for 27 days and plated failed to produce anycolonies on 7H11 plates. The reason to use rabbit serum is to see ifserum might facilitate the recovery of old dormant bacilli, however,serum alone had no effect on reviving dormant bacilli. Even afterprolonged incubation at 37° C. for up to 2 months, the dormant bacilliincubated with 7H9 medium or 7H9 plus rabbit serum produced no colonieson the 7H11 plates. Remarkably, in the presence of the peptides P2, P3,P4, dormant bacilli from the old culture became resuscitated and formedplenty of colonies. The degree of resuscitation correlated with peptideconcentrations, as seen by appearance of 235, 170 and 7.5×10⁵ CFU/ml at10, 5 and 1 μg/ml of the peptides, respectively. This is the firstdemonstration that dormant tubercle bacilli in cultures that do not formcolonies at all on plates could be resuscitated after appropriatetreatment in vitro.

Example 11 Resuscitation of Dormant TB Bacilli from Mouse Tissues bySpent Culture Supernatant of M. tuberculosis

[0089] Mouse spleen tissue samples (A, B, and C) derived from mice thathad been treated with antituberculosis drugs isoniazid and a newrifamycin derivative rifalazil did not give CFU on mycobacterial 7H11agar plates. A resuscitation experiment was set up as follows todetermine if the mouse tissues contained any dormant M. tuberculosisbacteria that could be resuscitated with the spent culture supernatantderived from M. tuberculosis. Tissue samples A, B, and C were inoculatedinto both fresh 7H9-ADC liquid medium and spent culture supernatant (in7H9-ADC) of M. tuberculosis H37Ra and incubated at 37° C. withoutshaking for 8 weeks. Samples A and B failed to grow in either 7H9 mediumor the spent culture supernatant. Interestingly, sample C, which failedto grow in fresh 7H9 medium, gave growth in the spent culturesupernatant. When the growth in sample C was plated on 7H11 plates, thegrowth showed typical features of M. tuberculosis and the identity of M.tuberculosis was confirmed by PCR sequencing of the pncA gene. This isthe first demonstration of resuscitation of dormant bacilli from in vivoderived tissues that were ostensibly sterile due to treatment by TBdrugs.

[0090] This example demonstrates that mammalian tissue that isostensibly free of M. tuberculosis may contain dormant forms of theorganism which are capable of resuscitation by culturing in M.tuberculosis ESPSN culture medium.

[0091] While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

REFERENCES

[0092] Cole, S. T., Eiglmeier, K., Parkhill, J., James, K. D., Thomson,N. R., Wheeler, P. R., Honore, N., Garnier, T., Churcher, C., Harris,D., Mungall, K., Basham, D., Brown, D., Chillingworth, T., Connor, R.,Davies, R. M., Devlin, K., Duthoy, S., Feltwell, T., Fraser, A., Hamlin,N., Holroyd, S., Hornsby, T., Jagels, K., Lacroix, C., Maclean, J.,Moule, S., Murphy, L., Oliver, K., Quail, M. A., Rajandream, M. A.,Rutherford, K. M., Rutter, S., Seeger, K., Simon, S., Simmonds, M.,Skelton, J., Squares, R., Squares, S., Stevens, K., Taylor, K.,Whitehead, S., Woodward, J. R., Barrell, B. G. (2001) Massive gene decayin the leprosy bacillus. Nature 409, 1007-1011.

[0093] Cole, S. T. Brosch, R. Parkhill, J., Gamier, T., Churcher, C.,Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S. Barry 3rd, C. E.,Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T.,Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin,N., Holroyd, S., Hornsby, T., Jagels, K. and Barrell, B. G. (1998)Deciphering the biology of Mycobacterium tuberculosis from the completegenome sequence. Nature 393, 537-544.

[0094] Dubos, R. J., and Davis, B. D. (1946) Factors affecting thegrowth of tubercle bacilli in liquid media. J. Exp. Med. 83, 409-423.

[0095] Dunny, G. M., and Leonard, B. A. B. (1997) Cell-cellcommunication in gram-positive bacteria. Ann. Rev. MicrobioL 51,527-564.

[0096] Heller D. N., Murphy C. M., Cotter R. J., Fenselau, C., and Uy,O. M. (1988) Constant neutral loss scanning for the characterization ofbacterial phospholipids desorbed by fast atom bombardment. Analyt Chem60, 2787-2791.

[0097] McCune, R. M., Feldman, F. M., Lambert, H. P., and McDermott, W.(1966) Microbial persistence. I. The capacity of tubercle bacilli tosurvive sterilization in mouse tissues. J. Exp. Med. 123, 445-468.

[0098] McKinney, J. D. Jacobs, W. R., and Bloom, B. R. Persistingproblems in tuberculosis, in Emerging Infections, R. Krause, J. I.Gallin, A. S. Fauci, Eds (Academic Press, New York, 1998), pp. 51-146.

[0099] WHO Report on the Tuberculosis Epidemic. (2000) GlobalTuberculosis Programme, World Health Organization, Geneva, Switzerland.

We claim:
 1. A supplemented medium for culturing mycobacteria,comprising a cell extract from Mycobacterium tuberculosis complex or atleast one substantially purified product from Mycobacterium tuberculosiscomplex, wherein said cell extract or said substantially purifiedproduct exhibits growth enhancement activity for said mycobacteria,initiation of growth for small inocula, or resuscitation activity fordormant mycobacterial bacilli, and a suitable culture medium.
 2. Thesupplemented medium of claim 1, wherein said mycobacteria is selectedfrom the group consisting of Mycobacterium tuberculosis complex,Mycobacterium paratuberculosis, and Mycobacterium leprae.
 3. Thesupplemented medium of claim 1, wherein said substantially purifiedproduct is selected from the group consisting of a component ofearly-stationary-phase culture supernatant, a component of stationaryphase supernatant, and a component of a cell extract.
 4. Thesupplemented medium of claim 1 wherein said substantially purifiedproduct is a phospholipid or a component of a phospholipid.
 5. Thesupplemented medium of claim 4, wherein said phospholipid or saidcomponent of a phospholipid is selected from the group consisting ofphosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine,phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid,arachidonic acid, and C18-C31 fatty acids with or without double bonds,and esters of C18-C31 fatty acids with or without double bonds.
 6. Thesupplemented medium of claim 1 wherein said substantially purifiedproduct is a protein or a fragment of a protein.
 7. The supplementedmedium of claim 6 wherein said protein or fragment of a protein isselected from the group consisting of protein Rv1147c, a peptidecorresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2,a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQID NO. 4 and a peptide corresponding to SEQ ID NO.
 5. 8. Thesupplemented medium of claim 1 wherein said suitable culture medium isselected from the group consisting of 7H12B, 7H9, 7H10, 7H11, Sauton'smedium, Dubos medium, egg-based medium, and Lowenstein-Jensen medium. 9.A method for reviving dormant bacilli of a mycobacterium species,comprising the step of exposing said dormant bacilli of saidmycobacterium species to cell extract of Mycobacterium tuberculosiscomplex or at least one substantially purified product of Mycobacteriumtuberculosis complex, wherein said cell extract or said substantiallypurified product exhibits growth enhancement and resuscitation activityfor mycobacteria, and wherein said substantially purified product ispresent in sufficient quantity to effect revival of said dormant bacilliof said mycobacterium species.
 10. The method of claim 9 wherein saidmycobacterium species is selected from the group consisting ofMycobacterium tuberculosis complex, Mycobacterium paratuberculosis, andMycobacterium leprae.
 11. The method of claim 9, wherein saidsubstantially purified product is selected from the group consisting ofa component of early-stationary-phase culture supernatant, a componentof stationary phase supernatant, and a component of a cell extract. 12.The method of claim 9 wherein said substantially purified product is aphospholipid or a component of a phospholipid.
 13. The method of claim12, wherein said phospholipid or said component of a phospholipid isselected from the group consisting of phosphotidyl-L-serine, dioleoylphosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine,tuberculostearic acid, arachidonic acid, and C18-C31 fatty acids with orwithout double bonds, and esters of C18-C31 fatty acids with or withoutdouble bonds.
 14. The method of claim 9 wherein said substantiallypurified product is a protein or a fragment of a protein.
 15. The methodof claim 14 wherein said protein or fragment of a protein is selectedfrom the group consisting of protein Rv1147c, a peptide corresponding toSEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptidecorresponding to SEQ D NO. 3, a peptide corresponding to SEQ ID NO. 4and a peptide corresponding to SEQ ID NO.
 5. 16. A method for thediagnosis of infection caused by a mycobacterium species, comprising,combining a sample for which the presence or absence of saidmycobacterium species is to be determined with medium supplemented withcell extract of Mycobacterium tuberculosis complex or at least onesubstantially purified product of Mycobacterium tuberculosis complex ina culture, wherein said cell extract or said substantially purifiedproduct exhibits growth enhancement and resuscitation activity formycobacteria; and analyzing said culture for the presence of saidmycobacterium species, wherein a finding of the presence of saidmycobacterium species indicates a positive diagnosis for said infection.17. The method of claim 16, wherein said mycobacterium species isselected from the group consisting of Mycobacterium tuberculosiscomplex, Mycobacterium paratuberculosis, and Mycobacterium leprae. 18.The method of claim 16, wherein said substantially purified product isselected from the group consisting of a component ofearly-stationary-phase culture supernatant, a component of stationaryphase supernatant, and a component of a cell extract.
 19. The method ofclaim 16 wherein said substantially purified product is a phospholipidor a component of a phospholipid.
 20. The method of claim 19, whereinsaid phospholipid or said component of a phospholipid is selected fromthe group consisting of phosphotidyl-L-serine, dioleoylphosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine,tuberculostearic acid, arachidonic acid, and C18-C31 fatty acids with orwithout double bonds, and esters of C18-C31 fatty acids with or withoutdouble bonds.
 21. The method of claim 16 wherein said substantiallypurified product is a protein or a fragment of a protein.
 22. The methodof claim 21 wherein said protein or fragment of a protein is selectedfrom the group consisting of protein Rv1147c, a peptide corresponding toSEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptidecorresponding to SEQ ID NO. 3, a peptide corresponding to SEQ ID NO. 4and a peptide corresponding to SEQ ID NO.
 5. 23. A kit for the diagnosisof infection caused by a mycobacterium species, comprising a sealedcontainer of medium supplemented with cell extract of Mycobacteriumtuberculosis complex or at least one substantially purified product ofMycobacterium tuberculosis complex, wherein said substantially purifiedproduct exhibits growth enhancement and resuscitation activity formycobacteria, in a carrier fluid.
 24. The kit of claim 23 wherein saidmycobacterium species is selected from the group consisting ofMycobacterium tuberculosis complex, Mycobacterium paratuberculosis, andMycobacterium leprae.
 25. The kit of claim 23, wherein saidsubstantially purified product is selected from the group consisting ofa component of early-stationary-phase culture supernatant, a componentof stationary phase supernatant, and a component of a cell extract. 26.The kit of claim 23 wherein said substantially purified product of saidmycobacterium species is a phospholipid or a component of aphospholipid.
 27. The kit of claim 26, wherein said phospholipid or saidcomponent of a phospholipid is selected from the group consisting ofphosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine,phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid,arachidonic acid and C18-C31 fatty acids with or without double bonds,and esters of C18-C31 fatty acids with or without double bonds.
 28. Thekit of claim 23 wherein said substantially purified product is a proteinor a fragment of a protein.
 29. The kit of claim 28 wherein said proteinor fragment of a protein is selected from the group consisting ofprotein Rv1147c, a peptide corresponding to SEQ ID NO. 1, a peptidecorresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3,a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding toSEQ ID NO.
 5. 30. A method for the treatment of infection caused by amycobacterium species in a patient in need thereof, comprising the stepof administering to said patient cell extract of Mycobacteriumtuberculosis complex or at least one substantially purified product ofMycobacterium tuberculosis complex, wherein said substantially purifiedproduct exhibits growth enhancement and resuscitation activity formycobacteria, and wherein said step of administering results in theamelioration of symptoms associated with said infection.
 31. The methodof claim 30 wherein said mycobacterium species is selected from thegroup consisting of Mycobacterium tuberculosis, Mycobacteriumparatuberculosis, and Mycobacterium leprae.
 32. The method of claim 30,wherein said substantially purified product is selected from the groupconsisting of a component of early-stationary-phase culture supernatant,a component of stationary phase supernatant, and a component of a cellextract.
 33. The method of claim 30 wherein said step of administeringresults in the revival of dormant bacilli of said mycobacterium speciesin said patient.
 34. The method of claim 30 wherein said substantiallypurified product of said mycobacterium species is a phospholipid or acomponent of a phospholipid.
 35. The method of claim 34, wherein saidphospholipid or said component of a phospholipid is selected from thegroup consisting of phosphotidyl-L-serine, dioleoylphosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine,tuberculostearic acid, arachidonic acid, and C18-C31 fatty acids with orwithout double bonds, and esters of C18-C31 fatty acids with or withoutdouble bonds.
 36. The method of claim 30 wherein said substantiallypurified product is a protein or a fragment of a protein.
 37. The methodof claim 36 wherein said protein or a fragment of a protein is selectedfrom the group consisting of protein Rv1147c, a peptide corresponding toSEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptidecorresponding to SEQ ID NO. 3, a peptide corresponding to SEQ ID NO. 4and a peptide corresponding to SEQ ID NO.
 5. 38. A pharmacological agentfor the treatment of an infection caused by a mycobacterium species,comprising, cell extract of Mycobacterium tuberculosis complex or atleast one substantially purified product of Mycobacterium tuberculosiscomplex, wherein said substantially purified product exhibits growthenhancement and resuscitation activity for mycobacteria, and aphysiologically suitable carrier.
 39. The pharmacological agent of claim38 wherein said mycobacterium species is selected from the groupconsisting of Mycobacterium tuberculosis complex, Mycobacteriumparatuberculosis, and Mycobacterium leprae.
 40. The pharmacologicalagent of claim 38, wherein said substantially purified product isselected from the group consisting of a component ofearly-stationary-phase culture supernatant, a component of stationaryphase supernatant, and a component of a cell extract.
 41. Thepharmacological agent of claim 38 wherein said substantially purifiedproduct of said mycobacterium species is a phospholipid or a componentof a phospholipid.
 42. The pharmacological agent of claim 41, whereinsaid phospholipid or said component of a phospholipid is selected fromthe group consisting of phosphotidyl-L-serine, dioleoylphosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine,tuberculostearic acid, arachidonic acid, and C18-C31 fatty acids with orwithout double bonds, and esters of C18-C31 fatty acids with or withoutdouble bonds.
 43. The pharmacological agent of claim 38 wherein saidsubstantially purified product is a protein or a fragment of a protein.44. The pharmacological agent of claim 43 wherein said protein or afragment of a protein is selected from the group consisting of proteinRv1147c, a peptide corresponding to SEQ ID NO. 1, a peptidecorresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3,a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding toSEQ ID NO.
 5. 45. A culture medium for culturing a mycobacteriumspecies, comprising isolated early-stationary-phase or stationary phasesupernatant of Mycobacterium tuberculosis complex.
 46. The culturemedium of claim 45 wherein said mycobacterium species is selected fromthe group consisting of Mycobacterium tuberculosis complex,Mycobacterium paratuberculosis, and Mycobacterium leprae.
 47. Theculture medium of claim 45 wherein said isolated early-stationary-phaseor stationary phase supernatant is sterile.
 48. The culture medium ofclaim 47 wherein said isolated early-stationary-phase supernatant issterilized by filtration.
 49. A method for reviving dormant bacilli of amycobacterium species, comprising the step of exposing said dormantbacilli of said mycobacterium species to isolated early-stationary-phaseor stationary phase supernatant of Mycobacterium tuberculosis complex,wherein said step of exposing causes revival of said dormant bacilli.50. The method of claim 49 wherein said mycobacterium species isselected from the group consisting of Mycobacterium tuberculosiscomplex, Mycobacterium paratuberculosis, and Mycobacterium leprae. 51.The method of claim 49 wherein said isolated early-stationary-phasesupernatant is sterile.
 52. The method of claim 51 wherein said isolatedearly-stationary-phase supernatant is sterilized by filtration.
 53. Akit for the diagnosis of infection caused by a mycobacterium species,comprising a sealed container comprising isolated early-stationary-phaseor stationary phase supernatant from a culture of Mycobacteriumtuberculosis complex.
 54. The kit of claim 53 wherein said mycobacteriumspecies is selected from the group consisting of Mycobacteriumtuberculosis complex, Mycobacterium paratuberculosis, and Mycobacteriumleprae.
 55. The kit of claim 53 wherein said isolatedearly-stationary-phase or stationary phase supernatant is sterile. 56.The kit of claim 55 wherein said isolated early-stationary-phase orstationary phase supernatant is sterilized by filtration.
 57. A methodfor the diagnosis of infection caused by a mycobacterium speciescomprising, combining a sample for which the presence or absence of saidmycobacterium species is to be determined with isolatedearly-stationary-phase or stationary phase supernatant of Mycobacteriumtuberculosis complex in a culture; and analyzing said culture for thepresence of said mycobacterium species, wherein a finding of thepresence of said mycobacterium species indicates a positive diagnosisfor said infection.
 58. The method of claim 57 wherein saidmycobacterium species is selected from the group consisting ofMycobacterium tuberculosis, Mycobacterium paratuberculosis, andMycobacterium leprae.
 59. The method of claim 57 wherein said isolatedearly-stationary-phase or stationary phase supernatant is sterile. 60.The kit of claim 59 wherein said isolated early-stationary-phase orstationary phase supernatant is sterilized by filtration
 61. A method ofinhibiting the growth of a species of mycobacterium comprising the stepof exposing said mycobacterium an isolated supernatant or cell extractfrom a culture of Mycobacterium tuberculosis complex that is at least 3months old, wherein said step of exposing has the effect of inhibitingthe growth of said species of mycobacterium.
 62. The method of claim 61wherein said mycobacterium species is selected from the group consistingof Mycobacterium tuberculosis complex, Mycobacterium paratuberculosis,and Mycobacterium leprae.
 63. The method of claim 61 wherein saidisolated early-stationary-phase or stationary phase supernatant issterile.
 64. The kit of claim 63 wherein said isolatedearly-stationary-phase or stationary phase supernatant is sterilized byfiltration